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目的:探讨bcl-2基因在阻塞性黄疸大鼠肝细胞凋亡中的调控作用。 方法:健康(?)Wistar大鼠,进腹,游离出胆总管,于肝门部结扎胆总管并切断,远端再予以结扎。进腹后不结扎切断胆总管作为阴性对照。结扎后3,7,14,21d处死大鼠,每组8只。采用胶原酶原位肝灌注法获取体外培养正常大鼠及阻塞性黄疸大鼠肝细胞。分别收取正常大鼠及胆总管结扎大鼠肝细胞1×10~9/L~(-1),用Trizol试剂盒一步法提取大鼠肝细胞总RNA,行RT-PCR扩增;用PCR扩增bcl-2,和对照β-actin基因片段;分离的正常大鼠及胆总管结扎14 d大鼠的肝细胞行原代培养,培养板内置小飞片,使用100μmol·L~(-1)甘氨鹅脱氧胆酸钠(GCDC)作用于两组肝细胞24h后,用FCM法分析肝细胞的凋亡比率,用TUNEL技术进行肝细胞凋亡的原位检测。 结果:体外培养正常大鼠及结扎3d大鼠肝细胞bcl-2 mRNA的表达为阴性,仅见β-actin条带;胆总管结扎大鼠体外培养肝细胞在结扎后7d即可见肝细胞内bcl-2 mRNA的表达,其吸光度A值为0.13±0.15;结扎后14、21d也可检测其表达,bcl-2 mRNA吸光度在结扎14d达高峰,为0.56±0.21,且与结扎7、21d相比,两者差异有显著性(P<0.05);100μmol·L~(-1)的GCDC作用正常大鼠肝细胞后,凋亡明显增加,凋亡率为34.9±2.9％;胆总管结扎14d大鼠的肝细胞凋亡比率为15
Objective: To investigate the regulatory effect of bcl-2 gene on hepatocyte apoptosis in obstructive jaundice rats. Methods: Healthy Wistar rats were infused into the common bile duct, ligated and cut off the common bile duct in the hilar and the distal end was ligated again. Into the abdomen after cutting off the common bile duct as a negative control. Rats were sacrificed 3, 7, 14 and 21 days after ligation, 8 in each group. In situ hepatic perfusion with collagenase was used to obtain hepatocytes in normal rats and obstructive jaundice rats. The normal rats and common bile duct livers were collected 1 × 10 ~ 9 / L ~ (-1) of rat liver cells, Trizol kit was used to extract the total RNA of rat hepatocytes, RT-PCR amplification; amplified by PCR The bcl-2 and control β-actin gene fragments were added into the culture medium. Primary hepatocytes isolated from the normal rats and the common bile duct were ligated for 14 days. Glycyrrhizic acid sodium deoxycholate (GCDC) was applied to the two groups of hepatocytes 24h later, the apoptosis rate of hepatocytes was analyzed by FCM, and the in situ detection of hepatocyte apoptosis was detected by TUNEL technique. Results: The expression of bcl-2 mRNA in normal rat and ligation-induced 3d rat liver cells was negative, only the β-actin band was observed. In cultured hepatocytes of common bile duct ligation rats, the expression of bcl- 2 mRNA expression, the absorbance A value of 0.13 ± 0.15; 14,21 days after ligation can also detect the expression of bcl-2 mRNA absorbance peaked at 14d, 0.56 ± 0.21, and compared with ligation 7,21 d, There was a significant difference between the two groups (P <0.05). After treated with 100μmol·L -1 GCDC, the apoptosis of hepatocytes in normal rats increased significantly, the apoptosis rate was 34.9 ± 2.9% The rate of hepatocyte apoptosis was 15